KCNH channel regulation by intracellular ligands

细胞内配体对 KCNH 通道的调节

• 批准号: 9896844
• 负责人: Tinatin I Brelidze
• 金额: $ 30.86万
• 依托单位: GEORGETOWN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9896844
• 关键词: Action Potentials; Affect; Affinity; Arrhythmia; Binding; Biochemical; C-terminal; Cardiac; Cell Proliferation; Cyclic Nucleotides; Defect; Deletion Mutation; Development; Disease; Docking; Electrodes; Electrophysiology (science); Epilepsy; Family; Future; Goals; HCN4 gene; Heart Abnormalities; Hippocampus (Brain); Homologous Gene; Human; Individual; Ion Channel; Knowledge; Length; Life; Ligand Binding; Ligand Binding Domain; Ligands; Link; Long QT Syndrome; Malignant Neoplasms; Membrane; Methods; Molecular; Molecular Mechanisms of Action; Mutagenesis; N-terminal; Oocytes; Pharmaceutical Preparations; Pharmacologic Substance; Pharmacology; Physiological; Property; Proteins; Publications; RNA Interference; Regulation; Research; Specificity; Structural Models; Structure; Surface Plasmon Resonance; Techniques; Tumor Tissue; Ventricular; X-Ray Crystallography; base; drug discovery; experience; neuronal excitability; novel; novel strategies; novel therapeutics; overexpression; patch clamp; screening; small molecule; small molecule libraries; tool; tumor progression; voltage clamp

Ion channels in the KCNH family perform diverse and important physiological functions, including the regulation of cancer progression, neuronal excitability and cardiac contraction. KCNH channels contain a Per-Arnt-Sim (PAS) domain in their N-terminal and cyclic nucleotide-binding homology (CNBH) domain in their C-terminal regions. These intracellular domains are the key functional domains that are largely responsible for the distinct properties of KCNH channels that determine their physiological applications. The goal of this proposal is to identify small molecule ligands that directly bind to the PAS and CNBH domains and uncover the mechanisms of their action on KCNH channels. We will identify and functionally characterize KCNH channel ligands using a novel strategy based on the combination of surface plasmon resonance (SPR) and electrophysiology as the principal methods. We will first identify small molecule ligands by screening libraries of small molecules against the PAS and CNBH domains of KCNH channels with the high-throughput SPR method. We will then determine the functional effects of the identified small molecule ligands with medium-throughput two- electrode voltage-clamp method. Finally to uncover molecular mechanisms of action of the identified ligands we will use a combination of patch-clamp current recordings, mutagenesis, X-ray crystallography, SPR and structural modeling. These studies will advance our knowledge of KCNH channel regulation by intracellular ligands and PAS and CNBH domains, provide pharmacological tools to study physiological contributions of KCNH channels and will greatly facilitate the development of novel pharmaceutical agents for treatment of cardiac arrhythmias, cancer and epilepsy.

KCNH家族中的离子通道执行多种重要的生理功能， 包括癌症进展、神经元兴奋性和心脏的调节 收缩。 KCNH 通道在其 N 端包含 Per-Arnt-Sim (PAS) 域 和 C 端区域的环核苷酸结合同源 (CNBH) 结构域。 这些细胞内结构域是主要负责的关键功能域 KCNH 通道的独特特性决定了其生理功能 应用程序。该提案的目标是鉴定直接作用的小分子配体 结合 PAS 和 CNBH 结构域并揭示其作用机制 KCNH 频道。我们将鉴定 KCNH 通道配体并进行功能表征 使用基于表面等离子共振 (SPR) 组合的新颖策略 和电生理学为主要方法。我们首先要识别小分子 通过针对 PAS 和 CNBH 结构域筛选小分子文库来筛选配体 使用高通量 SPR 方法对 KCNH 通道进行分析。然后我们将确定 已鉴定的小分子配体的功能效应，具有中等通量的双- 电极电压钳法。最终揭示其作用的分子机制 对于已识别的配体，我们将使用膜片钳电流记录的组合， 诱变、X 射线晶体学、SPR 和结构建模。这些研究将 增进我们对细胞内配体和 PAS 调节 KCNH 通道的了解 和 CNBH 领域，提供药理学工具来研究生理贡献 KCNH渠道的建立将极大促进新型药物的开发 用于治疗心律失常、癌症和癫痫的药物。